Pre-transplant accommodated organs resistant to anti-donor immunity

ABSTRACT

This invention includes the composition of organ grafts accommodated prior to transplantation and therefore resistant to rejection by preformed antibodies. Accommodation is achieved within the donor animal by administration of sub-lethal levels of accommodation inducing factors derived from animals sensitized to the donor.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a U.S. National Phase Application under 35 U.S.C. §371.Applicant claims the benefit of PCT/US01/02342 filed Jan. 25, 2001,published in English under PCT Article 21(2), which claims priority toU.S. Provisional Application No. 60/178,347, filed Jan. 25, 2000. Theentire contents of PCT/US01/02342 and 60/178,347 are incorporated hereinby reference.

The work described herein has been partially funded by Research GrantNo. R43 DK50737 from the U.S. government which may have certain rightsin this invention.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The field of the present invention relates to the transplantation oforgans and tissues, and particularly to the conditioning of a xenograftorgan while still in the donor to resist rejection antibodies in thegraft recipient (accommodation). The invention also relates to methodsfor assessing accommodation within the donor animal.

II. Background

A major barrier to the transplantation of organs from one mammalianspecies to another is rejection of the xenografts. Much of the rejectionis not related to tissue-specific antigens but results from therecipient being sensitized to the donor animal. For example, humans andOld World monkeys have circulating antibodies to the alpha galactosyloligosaccharide expressed on tissues in other animals, including pigs.The antibodies bind to any transplanted pig xenograft, bind complement,and destroy the graft within an hour. This rapid reaction is referred toas hyper-acute rejection (HAR). The graft is rapidly destroyed by thebinding of preformed natural antibodies to endothelial cells andfixation of the complement. Most of the preformed antibodies in humansand old world apes (>80%) are against Gal(alpha)1-3Gal epitopes(alphaGal).

Acute vascular xenograft rejection occurs at three to eight days posttransplant. Induced and recurrent anti-donor antibodies bind to theendothelium, leading to endothelial activation, small vessel thrombosis,and recruitment of macrophages and NK cells. Acute xenograft rejectionis also mediated by cellular rejection. In contrast to cellularallograft rejection, CD4+ cytotoxic lymphocytes contribute to the graftinjury.

The current methods for prevention of HAR target the binding of antibodyor the fixation of complement. Anti-donor antibodies or complement canbe depleted from the blood of the recipient. Hyper-acute rejection wasprevented in ABO mismatched cardiac allografts performed in baboons byinfusion of soluble trisaccharides of the A and B antigen to neutralizethe antibodies. Though the circulating antibodies persisted afterdiscontinuing the oligosaccharides, some grafts showed prolongedsurvival. Cooper D. K., et al., Transplantation 56: 769-77 (1993).Transgenic pigs which express human complement inhibitors or withreduced expression of alphaGal have been created.

These technologies are useful on a short-term basis; however, they arenot completely effective. Antibodies return rapidly after their removaland the procedure must be repeated frequently. Transgenic pigs arevariable in the level of expression of the transgene. With bothmethodologies, episodes of HAR and acute vascular rejection are common.Efforts to suppress acute xenograft rejection using conventionalchemotherapy have been only partially successful. In particular, theantibody response to pig xenografts has proved resistant to suppression.

To reliably achieve long-term survival of xenografts, immune toleranceor graft accommodation will be necessary. Immune tolerance involvesprogramming the recipient's immune system to be specificallyunresponsive to the graft. Accommodation refers to adaptation of thegraft to be resistant to an existing immune response.

Partial immune tolerance to pig xenografts has been induced by ablatingthe recipient immune system and reconstituting it in the presence ofporcine hematopoietic cells. Porcine hematopoietic cells are detectablea year later. This approach has three basic limitations. First,tolerance would not resolve the problem caused by pre-existing naturalantibodies. Additional efforts, such as removal of pre-existingantibodies by immune adsorption would be required. Second, the recipientis subject to a prolonged period of immune deficiency, putting it atrisk for opportunistic and zoonotic infections. Third, the tolerancewould be to antigens expressed on the hematopoietic cells only.Tolerance would not be induced to the tissue-associated antigens. Pigheart and kidney xenografts were fulminantly rejected in baboons usingthis protocol.

The transplantation of pig thymi into immune ablated recipients enhancestolerance as the recipient pre-thymocytes mature in the porcineenvironment. Using this approach, porcine skin graft survival ismarkedly prolonged in mice and modestly prolonged in primates. The basiclimitations described above with mixed chimerism would still be aproblem.

Patent application No. PCT/US94/05844 teaches the induction of immunetolerance of recipient lymphocytes to xenografts by infusing lymphocytesinto immune deficient donor animals. The tolerant cells are laterharvested and transferred back to the recipient, conveying tolerance tothe recipient. However, the preexisting immune response would limit theusefulness of that approach.

The mechanism of accommodation is unknown. It is not due to thedepletion of antibodies or to replacement of donor endothelium with hostendothelium within the graft. Immunohistochemistry of long term cardiacxenografts (hamster-to-rat) shows deposition of IgG, IgM, C3, and C6 onthe endothelium, but minimal fibrin formation.

The possibility has been explored that accommodated endothelial cellshave reduced expression of antigen. Though some reduction in antigenssuch as alpha gal was observed with accommodation, it was not thought tobe sufficient to protect the graft. Parker W. et al., Am. J. Pathol.152: 829-39 (1998).

It is known that accommodated grafts can be adoptively transplanted to asecond recipient. The factors responsible for accommodation are presentwithin the graft and do not require circulating regulatory cells orfactors. Miyatake showed that if rejection of a hamster heart graft canbe prevented in a rat recipient, the graft will also resist rejectionwhen re-transplanted into a second recipient identical with the firstrecipient. T. Miyatake, N. Koyamada, W. W. Hancock, M. P. Soares, and F.H. Bach. Survival of accommodated cardiac xenografts uponre-transplantation into cyclosporine-treated recipients. Transplantation65: 1563-1569, (1998). While the observation is of scientific value, itis not clinically useful. To apply this observation would require twoidentical subjects, such as human recipients, one who would host thedonor organ until accommodation is achieved. The organ would then beprocured and transplanted into the second subject. The very limitednumber of potential recipients with identical twins and the ethicallyunacceptable complications to the first recipient make this approachunfeasible.

Some success in achieving accommodation in cultured endothelial cellshas been reported. Dorling A., et al., Xenotransplantation, 5: 84-92(1998); and Dorling A., et al., Transplantation, 62: 1127-1136 (1996).Dorling et a. demonstrated that prolonged exposure in culture of porcineendothelial cells to normal human immunoglobulins produced endothelialcell changes suggestive of accommodation.

Apparent confirmation of these studies was provided by Shah et al.,Fifth Congress of the International Xenotransplantation Association,Abstract 199 (1999). Minimal resistance to complement mediatedcytotoxicity was achieved with 72 hours of culture. Better resistancewas observed with 120 to 144 hours of incubation. On the other hand,others. were unable to confirm these studies using primary endothelialcell cultures. McKane W. et al. Fifth Congress of the InternationalXenotransplantation Association, Abstract 200 (1999). They suggestedthat the apparent resistance reported by others may be an artifactrelated to the use of immortalized endothelial cells, whichconstitutively express anti-apoptotic genes.

In vitro culture is unlikely to have significant clinical utility.Accommodated endothelial cells would not have significant utility bythemselves. Furthermore, accommodation of cultured and transformedendothelial cells required a minimum of 72 hours and preferably 120hours of culture. See Dorling et al. (1996), supra. If the observationwere to be extended to ex vivo culture of whole organs maintained inculture, the organs would significantly deteriorate during this period.

Achieving accommodation within the recipient is very difficult, costly,and often ends in failure, with rejection of the graft.

Therefore, a need exists for a method of xenograft transplantation thatavoids the high costs, the complications, and the high risk of failureassociated with accommodation of the xenograft organ within therecipient after transplantation.

SUMMARY OF THE INVENTION

An objective of the invention is to provide a tissue or a graftaccommodated prior to transplantation of the tissue or graft.

A second objective of the invention is to provide a method foraccommodation of the donor graft prior to transplantation.

Another objective of the invention is to provide a method fordevelopment of improved in-donor accommodation technology.

In accordance with one embodiment of the invention, a method to producea tissue or organ accommodated in a donor mammal to resist rejection ina recipient mammal, is provided. The method comprises:

-   -   infusing a donor mammal at least one time with sub-lethal levels        of at least one accommodation-inducing factor; allowing        prolonged exposure to said accommodation inducing factor; and    -   harvesting one or more of the tissues or organs which are        accommodated.

In accordance with a preferred embodiment, the accommodation-inducingfactor is infused in a donor mammal which is in an immune deficientstate. In accordance with another preferred embodiment, theaccommodation-inducing factor is an antibody reactive with donorendothelium, such as pig endothelium, plasma cells, B lymphocytes, humanB lymphocytes, conditionally immortalized B lymphocytes, anti-alphaGalantibody, a cell expressing an accommodation inducing factor such as anantibody, a hybridoma comprising a cell expressing an accommodationinducing factor, T cells reactive with cells in the graft tissue ororgan, perforin, or Bandeiraea simplicifolia lectin. In accordance withanother preferred embodiment, the method further comprises the step of:determining that accommodation of the tissue or organ has been achieved,prior to transplantation of said organ or tissue.

In accordance with another embodiment, a xenograft organ or tissue isprovided. The xenograft organ or tissue is raised in a donor mammaltreated with an accommodation inducing factor. In accordance with apreferred embodiment, the xenograft organ includes, but is not limitedto a heart, a kidney, a liver, a lung, a pancreas, a heart-lungintestine, a spleen, or a thymus. The xenograft tissue includes but isnot limited to bone, skin, hair, eye, neural tissue, smooth muscle,skeletal muscle, cardiac muscle, myocytes, pancreatic islets,hepatocytes, embryonic stem cells, progenitor cells, or hematopoieticcells. In accordance with another preferred embodiment, the treatmentwith an accommodation inducing factor occurred while the donor mammalwas in an immune deficient state. In accordance with another preferredembodiment, the accommodation inducing factor is an antibody reactivewith donor endothelium, an antibody reactive with pig endothelium,plasma cells, B lymphocytes, human B lymphocytes, conditionallyimmortalized B lymphocytes, anti-alphaGal antibody, a cell expressing anaccommodation inducing factor, a hybridoma comprising a cell expressingan accommodation inducing factor, T cells reactive with cells in thegraft tissue or organ, perforin, or Bandeiraea simplicifolia lectin.

In accordance with yet another embodiment, a method for developingaccommodation factors is provided which comprises infusing a donormammal at least one time with sub-lethal levels of at least oneaccommodation-inducing factor; administering a tissue or cells from amammal other than the donor to the donor; allowing prolonged exposure tothe accommodation-inducing factor; and harvesting the accommodatedtissue or cells. In accordance with a preferred embodiment, theaccommodation-inducing factor is from an individual who is the intendedrecipient of said harvested tissue or cell. In accordance with anotherpreferred embodiment, the accommodation-inducing factor is infused inthe donor mammal which is in an immune deficient state. In accordancewith yet another preferred embodiment, the accommodation-inducing factoris an antibody reactive with donor endothelium, an antibody reactivewith a cell or tissue from a mammal other than said donor which wasadministered to said donor, a cell expressing an antibody reactive witha cell or tissue from a mammal other than the donor which wasadministered to said donor, an antibody reactive with pig endothelium,plasma cells, B lymphocytes, human B lymphocytes, conditionallyimmortalized B lymphocytes, anti-alphaGal antibody, a cell expressing anaccommodation inducing factor, a hybridoma comprising a cell expressingan accommodation inducing factor, T cell reactive to the tissue ororgan, perforin, or Bandeiraea simplicifolia lectin. In accordance withstill another preferred embodiment, the method comprises the additionalstep of determining that accommodation of said tissue or organ has beenachieved, prior to harvesting of the tissue or cell.

In accordance with another embodiment, the xenograft tissue or cell isan osteoblast cell, an osteo clost cell, skin, a skin epithelial cell, ahair follicle cell, eye cell, neural cells, a skeletal muscle cell, asmooth muscle cell, a cardiac muscle cell, pancreatic islet, ahematocyte, a stem cell, a progenitor cell, or a hemapoietic cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows accommodation in chimeric pigs by a lymphocyte lysis assay.72-5, 72-1, and 72-3 are chimeric pigs infused at pre-immune fetal stagewith bone marrow. The control sample is lymphocytes from an un-infusedpig. The lysis was evaluated by Trypan Blue—see Example 3 for detailedmethod—as a function of the concentration of sensitized sheep serum.

FIG. 2 shows accommodation of a chimeric pig (72-5) by a lymphocyteassay. The control is lymphocytes from an un-infused pig. The lysis wasevaluated by Trypan Blue—see Example 3 for detailed method—as a functionof the concentration of sensitized sheep serum. The sensitized sheepserum was isolated from a different sensitized sheep than the sheep inFIG. 1.

FIG. 3 shows protection of a chimeric pig heart from hyperacuterejection by human blood. The hearts of a control pig and chimeric pig(72-5) were perfused with sensitized human blood and the heart beat wasmonitored.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. Overview

The invention provides a method for production of an accommodated organor tissue suitable for transplantation and the accommodated organ.Accommodation is the process of conditioning an organ to resist injuryin a sensitized recipient. It is also referred to as adaptation. Theadaptation conveys resistance in the graft to cell death or apoptosis,procoagulation changes, and adhesion of leukocytes. In accordance withthe invention, the accommodation is achieved by infusion ofaccommodation inducing factor into a donor, allowing the graft to beexposed to the accommodation inducing factor, and harvesting the graft,accommodated while in the donor for resistance to rejection inrecipient.

An accommodation-inducing factor is any factor that causes accommodationwithin a donor of an xenograft organ or tissue. The factor can be analiquot of plasma from a mammalian species other than the donor.Alternatively, the factor can be a ligand purified from plasma orexpressed in an in vitro system. Preferably, the factor is an immunesystem component, for example a B lymphocyte or an antibody.

The accommodated organ or tissue is an organ or tissue which can resistrejection by a sensitized recipient. A sensitized recipient is anyorganism with preformed antibodies or memory T cells reactive with donorantigens, present in the graft-recipient prior to transplantation.Examples of sensitized organisms are humans and old world monkeys,sensitive to pig antigens. The sensitized recipient typically produces ahyper-acute and/or acute vascular xenograft immune response to donortissue, generally. The response is mediated by performed antibodies andT cells present in the recipient organism prior to the introduction ofthe xenograft. The organ or tissue graft is harvested from a chimericanimal.

In accordance with the invention, a chimeric mammal or animal is anymammal wherein an infused transgenic accommodation-inducing factorresides. For example, a piglet who receives a infusion of cells fromanother mammal is a chimeric animal. In a preferred embodiment, achimeric animal is a swine who received, during an immune deficientstage, an infusion of cells from another mammalian species, e.g., ahuman. The infusion occurs preferably when the donor is in an immunedeficient state such as a pre-immune fetus.

Factors that induce accommodation include but are not limited toantibody reactive with donor tissue, antibody reactive with donorendothelium, antibody reactive with pig tissue or endothelium in thecase where the donor is a pig or a member of the swine family, plasmacells, B lymphocytes, human B lymphocytes, conditionally immortalized Blymphocytes, anti-alphaGal antibody, a cell engineered to express anaccommodation inducing factor, a hybridoma comprising a cell expressingan accommodation inducing factor, and Bandeiraea simplicifolia lectin.The factor can be a natural, isolated factor, or it can be a cellengineered to express a ligand or the purified engineered ligand.

The accommodation factors may be derived from a member of a species thatwould be a xenograft recipient, e.g., a human or another mammal. In apreferred embodiment, the accommodation inducing factors are isolatedfrom an individual who will later become a recipient. The method ofdetermining, isolating, and manipulating each of the accommodationsinducing factors are well known to an artisan skilled in the art.

The invention also provides organ xenografts that are less susceptibleto rejection by preformed and developing immune elements, particularlyantibodies such as natural antibodies to alphaGal. These antibodies arepresent in most humans and Old World monkeys. The alphaGal antigen isexpressed on endothelial cells and other cells or tissues from mostother species, including pigs. Accommodation of a transplant organ canbe achieved within the organ donor with prolonged exposure of the graftto at least one accommodation inducing factor. The mechanism of antibodyaccommodation is not well understood. Without commitment to any onemechanistic explanation of the phenomena, it is believed thatendothelial and other cells exposed in the donor to accommodationfactors express agents that provide protection against antibodies in therecipient, leading to resistance.

The invention also provides for transplant tissues from a species otherthan donor, made resistant to preformed or developing antibodies madeagainst the tissue in a recipient. For example, human hematopoieticcells are placed into fetal pigs under conditions that expose them tosublethal concentrations of accommodation factors. These factors areproduced by cells infused into the fetal pig or could be produced by thepig or gilt/sow. The hematopoietic cells, such as granulocytes wouldresist destruction in a human host with antibodies against thegranulocytes.

The invention also provides a method and a kit for analysis anddetection of the level of accommodation within potential donor animals.The assays are performed on blood and tissues obtained from theprospective donor animal prior to transplantation of the donor organ.Analyses include detection of recipient accommodation inducing factors,for example, immunoglobulins, B cells, and T cells in the donor animal.Other analyses include in-vitro tests of resistance of circulating cellsand tissues taken from the prospective donor to lysis by reactiveantibodies and complement, or reactive cytotoxic T cells of therecipient. Other alternative analyses include assessment of blood andtissues from the prospective donor for over-expression of protectivegenes typically expressed within accommodated organs, such as hemeoxygenase-1, A-20, and bcl2. Bach, F. H. et al., Nature Medicine 3(2):196-204 (1997).

The invention also provides a method to asses methods and factors foraccommodation and improve the accommodation method according to theinvention.

A. Transplant Organs or Tissue is Accommodated Prior to Transplantation

The present invention induces accommodation of the transplant organ ortissue prior to transplantation, while the organ or tissue is still inthe donor animal. Induction prior to transplantation provides majoradvantages. The6 risk of rejection of the organ graft is significantlyreduced. The potential costs and complications to the patient related toefforts at preventing rejection are significantly reduced. Multipleattempts can be made at achieving accommodation in multiple donoranimals, if desired, and a donor which shows indication of besttransplantation results may be selected for donation of the xenograft.

A donor animal is a mammal. The mammal can be, for example, but notlimited to this example, a non-human primate, an artiodactyl, acarnivore, a rodent or a lagomorph. A pig fetus is a preferred recipientof the accommodation factor.

The accommodation factor is typically an antibody or an antibodyproducing cell but can include other factors that induce accommodationto include, but not be limited to: antibody reactive with donor tissue,antibody reactive with donor endothelium, antibody reactive with pigtissue or endothelium in the case where the donor is a pig or a memberof the swine family, plasma cells, B lymphocytes, human B lymphocytes,conditionally immortalized B lymphocytes, anti-alphaGal antibody, a cellengineered to express an accommodation inducing factor, a hybridomacomprising a cell expressing an accommodation inducing factor, T cellsreactive with cells in the graft tissue or organ, perforin, andBandeiraea simplicifolia lectin. The factor can be a natural, isolatedfactor, or it can be a cell engineered to express a ligand or thepurified engineered ligand.

The accommodation factors may be derived from any mammal, for example amember of a species that would be a xenograft recipient, e.g., a humanor another mammal. In a preferred embodiment, the accommodation inducingfactors are isolated from an individual who will later become arecipient. The method of determining, isolating, and manipulating eachof the accommodations inducing factors are well known to an artisanskilled in the art.

The process of accommodation requires prolonged exposure to theaccommodation inducing factors, at least 16 hours, up to 150 days. Atthat point, harvesting of the organ is undertaken.

In the preferred embodiment, a pig fetus is infused at an early stage ofdevelopment, preferably about 45 days. However, accommodation in anadult mammal is possible. The donor does not have to be in an immunedeficient state, but must be able to allow circulating accommodationinducing factors to persist in the donor for periods of time sufficientto allow accommodation of organs and tissues. However, infusion of animmune deficient mammal is preferred.

A first step in achieving an accommodated xenograft organ isintroduction of accommodation inducing factor into a donor animal.Preferably the immune components are introduced into donor at apre-immune stage of development. For example, cells are infused intodonor animals that produce sub lethal levels of ligand which bind tocells in the donor animal. The ligand induces accommodation. The cellsare infused under conditions which permit stable chimerism, i.e., stablepresence of recipient immune system components in the xenograft donororganism. Later, the accommodated organ for transplantation is harvestedand placed into transport medium under conditions suitable fortransplantation into a recipient animal.

As an example, B cells programmed to produce antibodies reactive withpig endothelium are infused into pre-immune fetal pigs. The low levelsof antibodies produced bind to the endothelial cells of the pig andinduce accommodation or resistance to complement dependent cytotoxicity.An organ such as a heart procured from the modified donor pig is thenplaced in transport medium, such as, for example, the University ofWisconsin solution and transplanted into a human recipient. In spite ofthe presence of antibodies reactive to pig in the human recipient,including anti-alphaGal antibodies, the organ is resistant tohyper-acute rejection.

Alternative embodiments utilize one or more sources of antibody, such asplasma cells and conditionally immortalized B lymphocytes. Cells areproduced which proliferate indefinitely in vitro, but are mortalizedbefore transplantation. For example, a thermolabile SV40 transformationgene can be inserted. The cells then proliferate at 33° C., but not at37° C. Further safeguards can be provided by other means, such as bysurrounding the transformation gene with loxP sites. The transformationgene can then be removed with Cre Recombinase. Nakamura, J., et al.,Transplantation 63(11): 1541-1547 (1997). The cells expressing theantibodies can be from the eventual xenograft recipient, but do not needto be from to the recipient. Accommodation does not depend on theinduction of tolerance in the recipient. Rather, it may depend on theprolonged expression of anti-apoptotic genes in the donor organ cells.

Additional alternative implementations would utilize T cells that arereactive with the donor animal. Perforin, the protein complex producedby cytotoxic T cells and responsible for cytolysis of target cells isstructurally quite similar to the membrane attack complex. Increasedresistance to cytotoxic cells is developed by exposure to perforin.

Another implementation of the invention includes the production oftissue or cells of a species different from the donor mammal. Forexample, human granulocytes are produced that are accommodated prior toinfusion into a human patient. They would then resist rejection anddestruction by preformed or developing antibodies present in therecipient. Human hematopoietic cells (preferrably 10⁷/pig, range 10¹ to10¹⁰ per pig) would be infused into preimmune fetal pigs at 45 daysgestation (range 12 days gestation to 7 days post-natal). Cells such asplasma cells, B lymphocytes, or hybridoma cells could be infused intothe fetal pig that produce accommodating factors. At a later time, forexample, after the birth of the pig, the human cells would be harvestedand prepared in a manner appropriate for transfusion or transplantationinto the recipient. The cells or the pig could be tested with assaysthat would predict accommodation within the human cells. For example,human cells could be tested for their resistance to cytotoxicity byantibodies or for the expression of genes known to protect cells fromprogrammed death. The pig could be tested for the presence of antibodiesreactive with the human cells.

B. Assays to Assess Donor Animals for Accommodation

The invention provides a method for assessing accommodation in the donoranimal prior to transplantation of the organ. The assay allowsconfirmation of accommodation prior to transplantation. It also providesfor a comparison of multiple donor animals infused with factors toaccommodate organs, and the selection of the best candidate. The assayimproves the results of transplant attempts and minimize the risk ofrejection of the xenograft by preformed immune components.

To be effective, the assay needs to be predictive of accommodation andneeds to preserve the organ tissue for transplantation. The ex vivoperfusion assay is considered the gold standard for predetermination ofhyper-acute rejection and resistance to rejection. In accordance withthe ex vivo perfusion assay, an organ, such as a heart, is perfused withserum or blood in a Langendorff apparatus. Non-accommodated organs ceasefunctioning after a few minutes. Accommodated organs function forseveral hours. However, the accommodated organ is less suitable fortransplantation after the procedure.

In accordance with the invention, an assay for accommodation includes ashort term ex vivo perfusion of tissue other than the transplant organ.Alternatively, accommodation is gauged by testing blood cells orendothelial cells obtained from the putative donor animal for resistanceto complement dependent cytotoxicity. Complement dependent cytotoxicityassays incubate the test cells with antibody known to be reactive andfresh complement. After a period of time, the viability of the cells isdetermined. With similar control cells, the antibody and complement killmost of the cells, while a larger proportion of accommodated cells wouldsurvive. Yet another method tests the donor animal for persistence ofthe infused cells, or tests the donor animal for antibodies or cellsreactive with the donor animal tissue. The methods involved are wellknown in the art.

C. Method for Enhancement of Accommodation within the Donor Animal

The invention provides a system for testing improvements andidentification of further accommodation stimulating agents. The systemconsists of infusion of various factors and cells into test animals andlater assessment of the modified animals for accommodation. As examplesof the system, multiple sources/fractions of pig reactive antibodies arecompared for their potential to induce accommodation. Each experimentalgroup would include fetal pigs infused with the particular fraction ofcells. Later the donor and/or the organs would be assessed foraccommodation. Examples of tests for accommodation would include the exvivo perfusion of an organ such as a heart or kidney and heterotopictransplantation of a xenograft into non-human primates. The cellfraction leading to the most prolonged function of the heart would beconsidered the most optimal. Additional objectives of the inventioninclude the testing of different strains of donor animals and oftransgenic animals.

II. Methods for Production of Accommodated Organs or Tissues in theDonor Animal

The present invention provides transplant organs or tissues accommodatedto be resistant to rejection by the recipient of the graft. It alsoprovides a method for production of such accommodated organs. Inaccordance with the method, the organ, before harvesting, is exposed ona repeated or chronic basis to accommodating factors which bind totissues in the donor animal but do not cause irreparable injury to theanimal or the organ. Following prolonged exposure, the accommodatedorgan is harvested and prepared for transplantation into a recipient,such as a human.

Preferably, the ligand either needs to be infused into the donor on arepeated basis or produced by cells that are stable within the donoranimal. The same or alternative or additional ligands are infused uponrepeat of the infusion step. The donor animal should preferably be in areduced immune activity state (immune deficient) in order to accept theligand, i.e., not to immunologically reject it.

As an example of ligand infusion, human immunoglobulins are infused intopre-immune fetal pigs. The immunoglobulins are injected into individualfetal pigs by percutaneous injection using ultrasound guidance.Sub-lethal levels of the accommodating factors are infused.

The levels of factor to be infused is pre-determined in empiricalfashion. For example, a serum fraction is used and increasingconcentrations are administered to at least one other fetus, at similarstages of development. An LCD₅₀ is determined and preferably used as anaccommodation treatment. Any LCD_(n), where n≦90% of the lethal dose isuseful as an accommodation factor dose.

The immunoglobulins include antibodies, including IgG and IgM, whichspecifically binds pig endothelial cells. Examples of such antibodiesinclude anti-alphaGal present in humans and Old World primates. Galili,U., et al., Blood, 82(8): 2485-2493 (1993). AlphaGal is not expressed inthese animals, but is expressed in most other species, including pigs.The immunoglobulins may include antibodies to other xenogeneic antigensas well.

The method of preparation of such antibodies or isolation of enrichedfractions of such antibodies are well known to artisans skilled in theart. For example, serum can be passed through an immunoadsorption columncontaining alphaGal. After washing the column, the adherentanti-alphaGal antibodies is eluted. Active antibody fragments or activesingle donor antibodies can also be employed.

Another example of a ligand would be the lectin Bandeiraea simplicifoliawhich binds to alphaGal, including the alphaGal expressed on pigendothelial cells. Grehan, J. F., et al., Transplant. Proc. 32(5):975(2000). The lectin is infused at low levels (less than 100 μg/ml) intoputative donor pigs. It is infused on a daily basis for 3 to 30 days.

B lymphocytes committed to production of antibodies or plasma cellsproducing antibodies reactive with the donor tissue are infused underconditions allowing stable engraftment and providing sub lethal doses ofantibodies. As an example, human B lymphocytes (2×10⁸/kg, range 1×10⁶/kgto 4×10⁹/kg) would be infused by ultrasound guidance into fetal pigs at45 days gestation (range 30 to 80 days) under sterile conditions. It isestimated that approximately 1% of the human B cells would be committedto production of antibody to alphaGal. Because the B cells are infusedinto pre-immune fetal pigs, the fetal pigs would accept the B cells andbecome tolerant to such cells.

The accommodation inducing factors, be they a ligand or a suspensioncontaining immune cells, can be from an individual unrelated to therecipient. Accordingly, the cells may include B cells from unrelatedhumans or from other species. For example, human B cells from a type Oindividual can be infused into fetal baboons of type A or B. The lowlevels of anti-A or anti-B induce accommodation. The transplant organcould then be harvested for transplantation after the birth of thebaboon. The infused cells may include immortalized cells such ashybridoma cells. In accordance with one embodiment, anaccommodation-inducing cell, preferably a B lymphocyte would contain asuicide gene such as thymidine kinase or adenosine deaminase. Cohen, J.L., et al., Hum. Gene Ther. 11(18): 2473-2481 (2000). Prior totransplant, the cells could then be purged with the appropriate prodrug.

In a preferred embodiment, the accommodation inducing factors are fromthe same species as the recipient. In accordance to another preferredembodiment, the accommodation inducing factor is from the individual whowill be the ultimate recipient. Because accommodation is a property ofthe transplant organ, it is not essential to adoptively transferlymphocytes back to the recipient.

Following prolonged exposure (preferably more than 20 days, range 16hours to 150 days), the donor animal is euthanized under conditions thatpermit procurement of the donor organ suitable for transplantation. Forexample, if the organ for transplant is a pig heart, the donor pig withaccommodated tissues is sedated and anesthetized with isoflurane. Understerile conditions, the chest is opened and cold Cardioplegia solution(such as Stanford solution containing mannitol, potassium chloride,sodium bicarbonate) is infused into the pig aorta and the heart ispacked in iced saline. The heart is removed and perfused with transportmedium, such as Eurocollins solution (Steffen, R., et al., Transpl. Int.3(3):133-136 (1990)) or University of Wisconsin solution (Belzer, F. O.and Southard, J. H. Transplantation 45(4): 673-676 (1988)) The organpreservation solutions contain essential amino acids, carbohydrates, andelectrolytes that maintain viability of the organ or tissue duringtransportation to the recipient.

Organs that may be accommodated are, for example, but not limited to fortransplantation prior to transplantation include, but are not limited toheart, kidney, liver, lung, pancreas, heart-lung, intestine, spleen, orthymus. Tissues that may be accommodated include, but are not limited tobone, skin, hair, retired pigmented cells, neural tissue, skeletalmuscle, myocytes, pancreatic islets, hepatocytes, embryonal or adultstem cells, and hematopoietic cells.

Another implementation of the invention relates to the production ofaccommodated tissue or cells of a species different from the donormammal. Such tissu or cells may include, but are not be limited to,osteo blast cells, osteo clost, skin, skin epithelial cells, hairfollicle cells, eye, neural cells, skeletal muscle, smooth muscle cells,cardiac muscle cells, pancreatic islets, hepatocytes, stem cells,progenitor cells, or hemapoietic cells. Such cells would be derived froman animal of a species other than the donor. Preferably, they would befrom the same species as an eventual recipient, or even from therecipient himself. The donor animal could be any of the donors of theinvention.

Accommodation factors in accordance with the invention could beadministered prior to administration of the cells for accommodation (onehour to two days), at the same time as the cells for accommodation orshortly after the cells for accommodation are administered. In apreferred embodiment, the cells for accommodation and the accommodationinducing factors are administered at the same time, mixed together, orwithin minutes of each other. The accommodation inducing factors arefactors in accordance with the invention, but may further include anantibody, an antibody producing cell, or a T-cell which recognizes cellsfrom the species where the cells to be accommodated are derived.

After prolonged exposure in accordance to the invention, the state ofaccommodation of the cells of a species different from the donor mammalcould be assessed by any of the methods of the invention, for example bylooking for antibodies in the donor specific for the cells introducedfor accommodation. Finally, the accommodated cells or tissue areharvested.

The cells introduced for accommodation or their progeny can be separatedby positive means or by negative means from donor cells. For example,the donor can be from a species engineered with a conditionally lethalgene, for example the thymidine kinase (HSV-tk) gene. Cells harboringthis gene are sensitive to ganciclovir. Moolten and Wells Cancer Res.46: 5276-5281 (1986); Reardon J. Biol. Chem. 264:19039-44 (1989); andPatil et al., Breast Can. Res. Treat. 62: 109-115. Upon harvesting,donor cells can be killed, by exposure to ganciclovir. Alternatively,the cells to be accommodated are transformed with a positive selection,prior the being administered into the donor. They can be engineered toexpress a selective marker, such as drug resistance, or an antigen thatwill upon expression be located on the cell membrane such that anantibody specific to the antigen may fish out a cell expressing theantigen. Those and similar approaches are well known to an artisanskilled in the art.

For example, human granulocytes could be produced that are accommodatedprior to infusion into a human patient. They would then resist rejectionand destruction by preformed or developing antibodies present in therecipient. Human hematopoietic cells (preferred 10⁷/pig, range 10¹ to10¹⁰ per pig) would be infused into preimmune fetal pigs at 45 daysgestation (range 12 days gestation to 7 days post-natal). Accommodationinducing factors such as plasma cells, B lymphocytes, or hybridoma cellscould be infused into the fetal pig that produce accommodating factors.At a later time, for example, after the birth of the pig, the humancells would be harvested and prepared in a manner appropriate fortransfusion or transplantation into the recipient. The cells or the pigcould be tested with assays that would predict accommodation within thehuman cells. For example, human cells could be tested for theirresistance to cytotoxicity by antibodies or for the expression of genesknown to protect cells from programmed death. The pig could be testedfor the presence of antibodies reactive with the human cells.

While pigs would be an ideal species donor mammal for accommodation, theinvention can be readily implemented with any of many animals. Animalsthat can potentially be used as surrogates i.e. donors, include, but arenot limited to those listed below. It is known that some animals offeradvantages for select uses.

Preferred donors are mammals. From among 39 major orders of the classMammalia, five orders appear particularly suitable as surrogate animalsfor human organ recipients: primates, artiodactyls, carnivores, rodents,and lagomorphs.

The primates, other than human, are suitable from the standpoint oforgan function. Amino acid sequencing of proteins typically demonstrate90 to 98% homology with humans. Organs such as livers and heartsfunction well when transplanted into humans. The primates are concordantwith humans, i.e. human recipients do not typically have performedantibodies to the tissues of the primates.

While some of the lower primates, such as lemurs, have short gestationperiods (132-134 days), the higher primates (chimpanzees, gorillas) havegestation periods approximating that of humans (267 days).

The artiodactyls, even-toed ungulates, include several domesticatedanimals such as swine, sheep, goats, and cows. Organs or proteins fromseveral members have been demonstrated to be functional and useful inhumans or have been proposed for transplantation. For example, porcineand bovine insulin, pig skin, sheep hearts, etc. have been used orproposed for therapeutic use.

The gestation periods vary between the members of this order. Pigs havea period of 114 days. Sheep have a period of 145 days. Cows have agestation period of 282 days. Cows offer features that are potentiallyuseful for surrogate tolerogenesis. The placental blood of all of thelittermates is shared. Therefore human cells infused into a single calfshould lead to tolerance to all of the littermates.

The carnivores, including dogs, cats, etc., have several features thatare potentially advantageous for surrogate tolerogenesis. Many haveshort gestation periods (cats about 65 days, dogs about 63 days) and thenewborn are relatively well developed. The canine and feline immunesystems are very similar to the human immune system. The felineimmunodeficiency virus model in cats is one of the few animal modelsavailable for the study of AIDS.

Cats and dogs have been commonly used as large animal models fortransplantation, including bone marrow, lung, intestine, and bonetransplants. Ladiges et al., Lab. Anim. Sci., 40: 11-15 (1990); andHenry, et al., Am. J. Vet. Res., 46: 1714-20 (1985).

The rodents, including rats, mice etc. are potentially useful forsurrogate tolerogenesis because of their short gestation periods andrapid growth to maturity. For example, rats have a gestation period ofonly 21 days and grow to maturity in only 6 weeks. Because the immunesystem of rodents is very immature at birth, tolerance can be induced byinjecting cells within 24 hours of birth rather than by intrauterineinjections.

The lagomorphs, which includes rabbits and hares, were once consideredpart of the rodent order but have been recently separated. They sharewith the rodents a very short gestation period and short maturationperiods. Thus, they would also be useful for the development of newdonor strains, including transgenic strains favorable providingfunctional organs or tissues. Their larger size would make these animalsbetter surrogate candidates than rodents.

The ideal surrogate species should preferably be phylogenetically closeto the intended organ graft recipient. Also, the physiology of theintended graft should be similar to the physiology of the organ graftrecipient organ or tissue to be replaced by the graft.

Additional considerations influence the choice of species for surrogatetolerogenesis. The transplanted graft is to be approximately the samesize as the corresponding graft within the organ graft recipient.

Consequently, with the additional considerations described above, pigsare preferable surrogates over primates, because pigs have a gestationperiod of only 114 days and typically grow to over 59 kg by four monthsof age.

Pre-immune fetal animals provide an ideal environment for infusion of Bcells or plasma cells. The earliest fetus that could be infused with anaccommodation inducing agent would be the first stage with a circulatorysystem and beating heart. The fetal development proceeds at differentrates in different species. However, this stage is known in the art asthe Carnegie stage 10 (4 to 12 somites). Human fetuses at this stage are2 to 3.5 mm in length. William J. Larsen, ESSENTIALS OF HUMANEMBRYOLOGY, Churchill Livingstone, p. xi (1998).

When the donor is a fetus, maternal factors may contribute toaccommodation. Many factors are transported from maternal circulatorysystem to the fetal circulatory system. For example, all four subclassesof IgG are transferred across the placental membrane. Coulam et al.,eds., IMMUNOLOGICAL OBSTETRICS p 295, WW Norton & Co. (1992). Placentaltransfer begins at the midpoint of the first semester (about 17 days inpigs) and continues at a low level through the second trimester(approximately 76 days in pig). In vitro, low levels of IgG were shownto accommodate porcine endothelial cells. Dorling et al., (1998), supra.Therefore, circulating IgG or other factors in the mother, eitherintroduced by infusion, or produced by the mother, would be transferredacross the placenta to the fetus, leading to accommodation in the fetus.

There are alternative ways in which an accommodation-inducing factor,for example, B cells or plasma cells, may achieve stable engraftment andproduce anti-donor antibodies. For example, B cells may be infused intoanimals that are immune deficient due to chemotherapy or irradiation orare immune deficient due to congenital defect such as severe combinedimmune deficiency. The mammal may receive bone marrow or stem celltransplants, similar to the method used for human bone marrowtransplants. Armitage, J. O., Blood 73(7): 1749-1758 (1989). Mammalswith a congenital immune deficiency such as severe combined immunedeficiency can be infused with foreign cells, such as humanhematopoietic cells. McCune, J. M., et al., Immunol. Rev. 124: 45-62(1991).

Stable engraftment could also be achieved by infusing cells into thedonor animal that are not antigenically recognized by the donor animal.As an example, porcine B cells could be transfected with a geneproducing an antibody reactive with porcine endothelial cells. Thesecells could then be infused into pigs to be used as donor animals.

Under specific circumstances, the factors may be administered by anyroute known to a skilled artisan, as, for example, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcuterion, intraperitoneal, sublingual,or rectal. The preferred manner of administration of the accommodationinducing factors is by infusion.

The factors are delivered in pharmaceutically accepted formulations.They can be aqueous, preferably in a physiologically compatible buffer,e.g. Hank's solution, Ringer's solution or buffered saline.Alternatively, powders, gels, emulsions may be administered. Theformulations depend on avenue of delivery and are well known to anartisan skilled in the art. For a discussion of formulations andadministration methods, see REMINGTON'S PHARMACEUTICAL SCIENCES (MaackPublishing Co., Easton, Pa.)

III. Methods for Assessment of Transplant Organ Accommodation Prior toTransplantation

After the above procedures have been performed, the optimal utilitywould be achieved by analyzing the donor pigs or tissues from donor pigto determine if accommodation has been achieved and to quantify theaccommodation. In general the test should be relatively rapid andcost-effective. It should be predictive of the graft survival. It shouldnot interfere with the organ to be transplanted.

Analysis of the donor animal for predictive evidence of accommodationwould achieve three basic goals. First, the analyses provides qualityassurance and confirmation of accommodation. If accommodation is notachieved in the tested donor animal, the transplant could be postponedor another animal could be used. Second, tests of accommodation canconfirm if the graft is specifically accommodated for a particularpatient. Third, the analyses permit the comparison of multiple potentialdonor animals and the selection of the most suitable animal fortransplantation.

The analyses of accommodation can be accomplished via any of four basicapproaches: in vitro assays of rejection, detection of genes in tissuethat convey protection against rejection, detection of either cells orimmunoglobulins infused in the donor animal prior to organ harvestingand detection of antibodies in the test animal reactive with the donoranimal.

In the first approach, tissue from the donor animal is assessed invitro. Plasma or serum from the recipient, or a sensitized animalsimilar to the recipient, is infused through tissue from the testanimal. If accommodation was not achieved, the tissue would ceasefunctioning shortly after perfusion. If accommodation is achieved,however, the tissue continues to function. As an example, in theLangendorff assay, heart grafts that are perfused with plasma, serum, orblood containing cytotoxic antibodies stop beating in about 20 minutes.If accommodation is achieved, however, the graft would beat for two tofour hours. Therefore the heart graft obtained for transplantation couldbe perfused with sensitized plasma. If it were still beating strongly atthe end of 45-60 minutes, accommodation would be demonstrated. The heartgraft could then be stopped with cardioplegia and perfused withtransport medium until transplanted. See Examples 2 and 4.

In an alternate and preferable embodiment, another organ from the donoranimal is tested. For example, if the transplant organ were a kidney,the heart or the contralateral kidney could be tested for function withthe Langendorff assay. Accommodation would be considered to be achievedif the test organ continues to be considered viable for at least a 50%longer time interval, preferably at least a 100% time interval, than thetypical time an equivalent organ which did undergo anaccommodation-inducing step is deemed viable.

In another implementation of the in vitro test of rejection, peripheralblood leukocytes or endothelial cells from the chimeric donor mammal isincubated with serum containing antibodies which specifically bind todonor mammal tissue and complement. Complement mediated lysis of cellsfrom the donor is determined. If accommodation is achieved, lysis issignificantly reduced compared to lysis of a similar unmodified animal.See also Example 3.

Viability can also be determined with other dyes. For example, deadcells will accumulate propidium iodide and can be detected by flowcytometry. Cells can be labeled with radioactive Chromium-51. Whenkilled, the radioactivity is released. After removing the cells andmembranes, the free radioactivity is measured. Shimizu, Y., et al., J.Immunol. Methods 164: 69-77 (1993). See Example 5.

The second basic approach of analyses for accommodation would testtissue from the modified animal for expression of genes that protecttissues from hyper-acute rejection. These gene products make cellsresistant to cellular death by apoptosis and prevent expression ofproinflammatory genes including cytokines, procoagulant and adhesionmolecules. Examples of these genes include heme oxygenase, A-20, andBcl-2. They have been observed in grafts that become accommodatedfollowing prolonged transplantation in a sensitized recipient.

In accordance to the present invention, in one embodiment, a biopsy istaken from the test donor animal, such as a pig modified as describedabove to induce accommodation to preformed anti-pig antibodies. Thebiopsy could be from the transplant organ, such as needle biopsy of theleft ventricle of the heart. Alternately, it could be from another organor tissue. If the transplant organ were to be the heart, it could be abiopsy from the kidney, skin, or a sample of blood. The tissue isexamined for the presence of the above genes using standardantibody-based assays such as immunohistochemistry, Western blots, andELISA assays, and RNA using Northern blots and polymerase chain reactionassays. The results are compared with positive and negative controls.Monoclonal antibodies for Bcl-2 can be obtained from TransductionLaboratories (Lexington, Ky.) and for heme oxygenase from StressGen(Victoria, BC, Canada). Monoclonal antibodies against A-20 were preparedby Dr. V. Dixit (Ann Arbor, Mich.). A probe can be devised or primerscan be devised and purchased or synthesized on a DNA synthesizer bymethods well known in the art. Because others have not accommodated anorgan while still in the donor, there was no reason to have tested thedonor organ for enhanced expression of the gene prior totransplantation. Neither was there any reason to test tissues from thedonor animal other than the transplant organ for evidence ofaccommodation.

The third approach to detection and quantification of accommodationassesses evidence of chimerism within a donor animal with infused cellsor which was treated with an accommodation-inducing factorimmunoglobulin. These parameters can be readily assayed and provideindirect evidence of accommodation. If the donor animal rejected theinfused cells and the immunoglobulins were undetectable, it is unlikelythat accommodation of an organ or tissue was achieved. Similarly, theirpersistence is predictive of accommodation.

A sample of anticoagulated blood or serum is obtained from the modifieddonor animal, such as a pig infused as a fetus with B cells. Theperipheral blood lymphocytes are stained for chimeric lymphocytes andthe number of cells quantified with analytical flow cytometry. As anexample, if the pigs were infused with human B cells, the lymphocytesfrom the modified pig could be stained for human B lymphocytes with anantibody specific to CD20. The CD20 is specific of the species providingthe accommodation factor. The amount of chimerism would be compared withappropriate positive and negative controls. The lymphocytes arealternately assayed for DNA present only in the infused cells. The DNAis detected by polymerase chain reaction or restricted length fragmentpolymorphisms.

In accordance with an alternative embodiment, the cells infused into thedonor animal are labeled with a marker that is readily detected. Forexample, the infused cells may contain a transgene containing greenfluorescent protein (GFP). Blood from the modified donor animal isexamined directly for GFP by fluorescence microscopy or by flowcytometry.

In another embodiment, the serum is assayed for immunoglobulins producedby the infused cells. Human immunoglobulins, for example, could bequantified using standard assays such as an ELISA assay. See Example 6.

The fourth approach to analyses of the modified donor animal foraccommodation is by detection of antibodies specific to the donor tissueor to cells producing antibody which specifically binds the donortissue. Serum or anticoagulated blood is obtained from the donor animalpretreated with an accommodation-inducing factor, such as a pig infusedwith human B cells reactive with pig endothelial cells. The serum is cocultured with porcine endothelial cells. The cells are washed andlabeled with peroxidase-conjugated anti-human IgG and IgM, followed withthe appropriate substrate. The presence of low levels of anti-pigantibody is a positive result. The values are compared with appropriatenegative and positive controls. Alternatively, lymphocytes obtained fromthe modified donor animal are maintained in culture. The spentsupernatant is assayed for production of anti-donor antibodies.Alternatively, immunoglobulins are isolated from one or more of tissuesfrom the modified donor animal. For example, if the transplant organ isa heart, a kidney is removed from the modified animal and perfused withan acidic buffer (pH2 to pH4) such as glycine buffer. The antibodiescollected in the eluate are assayed for anti-donor antibodies.

IV. Methods for Improvement or Accomodation Procedures

The present invention provides an experimental system for testingimprovements to the donor organ accommodation and, in particular, thetesting of accommodation factors. In particular, the invention allowsfor quantification of accommodation.

As an example of implementation of the system, multiple sources ofanti-pig antibody are assayed to develop optimal accommodation of pigxenografts. Potential sources of antibodies include B cells isolatedfrom an unsensitized human, B cells isolated from a sensitized human, Bcells isolated from sheep, a B cell line known to produce antibodies toalphaGal, liposomes containing Bandeiraea simplicifolia, etc. Litters ofpre-immune fetal pigs are infused with increasing doses of the antibodyor cells expressing antibody using ultrasound guidance, until thetoxicity causes abortion of the fetal pigs. Using a sub lethal dose ofthe test source, such as 50% of the lethal dose, fetal pigs are infusedwith the test source. After birth of the pigs or Caesarian section ofthe sows, the test pigs are assayed for accommodation using any of theabove described tests. At various ages, heart grafts are harvested fromappropriate pigs and tested by the Langendorff assay, perfusing thegraft with sensitized plasma or blood, such as human blood. The optimalantibody or antibody producing cell is the one that provides the mostprolonged function in the Langendorff assay and provides for persistenceof accommodation after birth. A minor population of the testedaccommodation inducing material is further tested withxenotransplantation into sensitized recipients, such as baboons.

The system can also be used to test sources of animals foraccommodation, including different strains of pigs, transgenic pigs, orother mammals.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1

Prevention of HAR in Sensitized Sheep Using Chimeric Pigs

Methods. Chimerism in three donor pigs was induced by infusing asuspension of lymphocytes obtained from a sheep (either the recipientsheep or an unrelated sheep) into pre-immune fetal pigs at 45 daysgestation. The suspension includes B cells programmed to produceantibodies reactive with pig cells. Later the pig heart was procuredfrom the donor and a heterotopic pig heart transplant was performed

Results. The results are presented in Table 1. The chimeric grafts wererejected at 3+, 9+ and 25 days in sensitized recipients compared withvascular rejection at 0.5-2 days for the non-chimeric controls.

The prolonged survival of the grafts was not due to tolerance. Indeed,the second sensitized sheep demonstrated a titer of cytotoxic antibodiesto pig at greater than 1 to 2000 by seven days post-transplant, yetcontinued to function appropriately.

The third experimental transplant was a sheep unrelated to the sheepused for chimerism. It was intentionally sensitized with pig lymphocytesthree times prior to the transplant.

Conclusions. Chimerism appears to provide protection against hyper-acuterejection in sensitized sheep. The third recipient suggests that theaccommodation is not a function of an individual sheep. It is highlyunlikely that the observed prolongation of graft survival was due toinduction of tolerance. Indeed, one of the recipients demonstrated atypical second set sensitization. Second set sensitization refers to theimmune reaction following a repeated exposure to the antigen. It istypically more rapid, higher tittered, and involves memory cells andIgG.

TABLE 1 Heart Xenograft Survival In Sensitized Recipients Pre-txAnti-Xenograft Experimental Donor Pig Pig Survival Xenograft Group ChimerismAntibodies (Days) Pathology Control* No 64 0.5 Severe vascular rejectionControl* No 4 1 Severe vascular rejection Control No 4 2 Severe vascularrejection Chimeric Yes 16 9+ No rejection Experimental Chimeric Yes 4 25Mild cellular Experimental rejection, ischemic infarct Chimeric Yes 163+ Mild rejection, Unrelated hematoma Experimental* *Sheep sensitizedwith pig lymphocytes three or more times. +Sheep euthanized fortechnical reasons. Graft rejection was absent to mild. The pre-tx standsfor prexenograft transplantation.

Example 2

Function of Chimeric Sheep to Pig Heart in Langendorff with SensitizedSheep Plasma

In this study, chimeric pig hearts were assessed for function in theLangendorff apparatus and compared with non-chimeric hearts. Previousstudies showed this assay to be predictive of in vivo function.

Sheep bone marrow was prepared and infused into fetal pigs at 45 daysgestation. At term, the pigs were delivered by Cesarean section. Bloodwas analyzed for chimerism by polymerase chain reaction employingprimers specific for class I histocompatiblity antigen, and flowcytometry. Heart grafts were isolated from chimeric or non-chimeric pigsand suspended in a Langendorff apparatus. The coronary arteries of thegraft were perfused with Krebs-Heinsleit solution. After beating for 30minutes, plasma from unsensitized sheep or sheep sensitized 3 to 5 timeswith pig lymphocytes was added. Neither the sheep nor the piglymphocytes used for sensitization were related to the sheep or pig inthe chimeric hearts. Complement dependent lymphocytotoxicity assaydemonstrated that the sensitized sheep plasma had titers of 32 to 128.The graft was analyzed for heart rate, perfusion pressure, and coronaryflow rate until the graft stopped beating (<30 beats/minute).

Results: Normal control hearts perfused with plasma from unsensitizedsheep functioned as long as four hours. Normal control hearts perfusedwith plasma from highly sensitized sheep stopped beating at 19+/−12minutes (7 to 40 minutes, n=6). These included 2 pigs with minimalchimerism (<1%) and 1 pig from an injected sow with no detectablechimerism. Hearts from chimeric pigs (3 to 11% by flow cytometry) beatfor 183+/−46 minutes (137 to 229 minutes, n=3). The difference wassignificant, P<0.0001. Immunofluoesence stains demonstrated less IgG andIgM deposition in the vessels of the chimeric hearts.

Conclusions: In the presence of high titers of cytotoxic anti-pigantibody, normal pig hearts stopped beating in less than 20 minuteswhile the chimeric hearts continued beating for 3 hours. Resistance torejection depended on factors within the heart graft rather thancirculating factors or cells. The protective effect was not specific fora particular sheep or particular pig. The sheep used for plasma for theperfusion was sensitized with pig lymphocytes not related to the pigsource of the heart. That sheep was also unrelated to the sheep whosecells were infused into the fetal pig.

One pig with minimal chimerism and 2 pigs from injected sows but nodetectable chimerism showed no evidence of accommodation and the heartgrafts stopped beating shortly after adding the plasma from a sensitizedsheep. These observations demonstrate the value of analyses of pigsprior to organ procurement.

Example 3

Accommodation of Leukocytes in Chimeric Pigs

Methods: Peripheral blood lymphocytes were obtained from normal pigs andfrom chimeric pigs injected with sheep marrow at the pre-immune fetalstage. The lymphocytes were incubated with tittered serum from asensitized sheep and with human serum containing cytotoxic antibodies toalphaGal. Fresh rabbit complement was added. Cytotoxicity was determinedwith Trypan blue. The titer was determined at the greatest dilution with20% or more cytotoxicity.

Results: The peripheral blood of three test pigs showed chimerism withsheep cells, varying from 2.7% (72-3) to 4.6% (72-5).

Peripheral blood lymphocytes from the three chimeric pigs have at leastpartial resistance to lysis by a sensitized serum. At a dilution ofserum causing 100% lysis of control pig lymphocytes, it caused lysis ofonly 11%, 31%, and 47% of the lymphocytes from the chimeric pigs. SeeFIG. 1.

The accommodation of pig 72-5 was confirmed by lysis of lymphocytes withserum from a second sensitized sheep. At a dilution causing 90% lysis ofnormal pig lymphocytes, the serum caused only about 15% lysis of thelymphocytes from the chimeric pig. See FIG. 2.

Conclusions: The peripheral blood lymphocytes from the chimeric pigsdemonstrated resistance to complement mediated cytotoxicity. Thisprotection cannot be accounted for by the presence of sheep lymphocytes,since the chimerism was less than 5%. The heart from piglet 72-5,demonstrating the greatest accommodation of lymphocytes, was tested foraccommodation in an ex vivo perfusion study using human blood.

Example 4

Prolonged Function of a Pig Heart Accommodated by Sheep Factors whenPerfused with Human Blood.

Methods: Pre-immune fetal pigs were infused with sheep marrow asdescribed in Example 3. After birth, the blood is analyzed by PCR andflow cytometry for chimerism with sheep cells. Accommodation wasassessed by measuring the resistance to complement dependent antibodycytotoxicity, as illustrated in Example 3. The heart explants from thebest chimeric pig (72-5) and a control pig were perfused ex vivo withblood from a human volunteer (type B) using the Langendorff apparatus.J. J. Dunning, et al., Eur. J Cardiothorac Surg. 8:204-206, (1994). Theduration of heart function was determined. Sections of the heartexplants were fixed in formalin and evaluated with routine hematoxylinand eosin sections.

Results: Consistent with the studies by Dumming, Id., the control pigheart became bradycardic within 20 minutes of perfusing it with wholehuman blood. The ventricles stopped at that time. When human blood fromthe same volunteer was perfused through a heart graft from a chimericpig (injected with sheep marrow), it initially demonstrated some slowing(from 132/minute to 70/minute). By 20 minutes, however, the raterecovered to 80% of the initial rate. By 80 minutes, the rate recoveredto 100% of the initial rate. The heart continued to beat strongly for 4hours, when the assay was terminated. See FIG. 3.

The histology of the control graft showed evidence of hyperacuterejection, including thrombi of platelets and fibrin, margination ofneutrophils, and interstitial hemorrhage. The myofibrils had evidence ofischemia, with hypereosinophilic cytoplasm, pyknotic nuclei, andvacuolization. In contrast, sections of the experimental heart did notshow thrombi, neutrophil margination, or ischemia. Immunofluorescencestains were done for human IgG, IgM, C3, and C9. The heart from thechimeric pig had less deposition of IgM, C3, and C9 than the controlheart. There was no appreciable difference in IgG deposition.

Conclusions: The study demonstrates prolonged function of a sheep factorchimeric heart when exposed to human blood. Because sheep constitutivelyproduce alphaGal, they do not develop natural antibodies to alphaGal, incontrast with humans. Therefore, the protection of the graft byaccommodation to anti-alphaGal was produced by antibodies to pigassociated antigens other than alphaGal.

Example 5

Persistence of Accommodation in Chimeric Pigs

Introduction: In this study, accommodation was assessed in 12 and15-week-old swine.

Methods: Peripheral blood lymphocytes were isolated from accommodatedand control swine. The lymphocytes were incubated with serum from asensitized sheep (titer 1:128) and fresh rabbit complement. Thesensitized serum was used at a dilution of 1:4. The killed lymphocyteswere quantified with propidium iodide and flow cytometry. The sensitizedserum and complement killed 96 to 100% of pig lymphocytes from eightcontrol pigs with these conditions. The viability of the controllymphocytes, therefore, was equal to or less than 4%. By contrast, 73%or more of lymphocytes from accommodated swine were viable. See Table 2.

TABLE 2 Viability at Current Current Swine Current Swine Pig 2 wks*Viability* age weight (kg) 421-3 83% 79% 12 weeks 77 421-10 80% 84% 12weeks 74 2160-2 90% 83% 12 weeks 78 2163-1 87% 90% 12 weeks 80 2141-279% 73% 15 weeks 94 *Control lymphocytes (n = 8) had viability equal toor less than 4%.Discussion: The accommodation of the lymphocytes observed intwo-week-old pigs persisted until tested at 12 or 15 weeks of age. Atthis age and weight, the swine would be appropriate donors for adulthuman recipients. Assuming that endothelial cells have a similar levelof protection, the accommodated organs would provide protection againsthyperacute rejection and acute vascular rejection.

Example 6

Presence of Sheep IgG in Accommodated Chimeric Pigs and TransplantationResults

Introduction: Accommodation is believed to result from the induction ofprotective proteins in cells with bound immunoglobulin. A possiblemechanism of accommodation in the chimeric pigs, therefore, would be theproduction of sublethal levels of antibodies by the infused xenogeneiccells. In this study, serum samples from donor pigs were analyzed byELISA for sheep IgG.

Methods: A sandwich ELISA assay was developed for sheep IgG. Themicrotiter plates were coated with donkey anti-sheep IgG, blocked withTween 20 and washed. The test serum was added at multiple dilutions andincubated for 30 minutes. After washing, biotinylated donkey anti-sheepIgG was added to the plates. After washing, the plates were developedwith streptavidin peroxidase, followed by substrate. The reaction wasstopped with sulfuric acid and the plates were read with a microplatereader. The assay was performed with serum from four control sheep, 14control pigs, and 12 pigs (10 to 14 days) from sows carrying pigsinjected with sheep marrow cells. Hearts were harvested and transplantedinto recipient sheep. Post-transplant, the sheep received cyclosporine(300-800 ng/ml trough level) and tapered steroids. The hearts werebiopsied weekly. See Table 3.

Discussion: The presence of chimeric IgG in the chimeric pigs wasdetected using a specific ELISA assay. The findings are significant forthree reasons. First, the presence of IgG is consistent suggests thatlow levels of antibodies bind to pig cells and induce accommodation.Second, the assay provides the means to monitor efforts at improvingaccommodation by enhancing B cell chimerism and function. Third, theassay is useful for quality assurance, assessing potential donor pigsprior to procuring the organ for transplantation.

This study does not define the specificity of chimeric IgG in predictinggraft outcome. However, none of the heart xenografts from pigs withdetectable IgG developed apparent rejection during the course of thestudy. Two sheep, however, were euthanized at 5 and 17 days due totechnical failures. Two sheep were euthanized at 30 days according tothe protocol. One graft was free of rejection after 106 days.

TABLE 3 Sheep IgG Recipient Graft Pig (μg/ml) Sheep Survival Outcome2141-4 0.64 105  5 days No rejection, technical failure 395-4 1.00 106106 days No rejection 1187-5 1.08 116  17 days No rejection, technicalfailure 2165-5* 2.16 110  30 days No rejection, euthanized 2161-2* 4.6497  30 days No rejection, euthanized 1115-7# Not 101  6 days Acutevascular detected rejection 2141-8# Not 112  7 days Acute vasculardetected rejection 1115-5 Not 113  5 days No rejection, detectedtechnical failure 2126-5 Not 103  9 days Acute cellular detectedrejection 2165-3 Not 115  15 days Acute cellular detected rejection385-1 Not 102  24 days Acute cellular detected rejection 1163-3 Not 107 25 days Acute cellular detected rejection Control Not pigs detected (n= 14) *Lymphocytes from pigs 2165-5 and 2161-2 were also tested with thelymphocytotoxicity assay for accommodation. See Example 5. Usingconditions in which the viability of control lymphocytes was equal to orless than 4%, the viability of 2165-5 lymphocytes was 98%. The viabilityof 2161-2 lymphocytes was 95%. #Control transplants. Pigs did not havedetectable chimerism by PCR or flow cytometry.

1. A method for providing a donor mammal tissue or organ resistant tocomplement-dependent cytotoxicity mediated by preformed antibodiesreactive with donor antigen in a sensitized recipient mammal of aspecies other than the donor mammal, comprising: infusing a non-humanpre-immune fetal donor mammal at least one time with sub-lethal levelsof bone marrow; allowing prolonged exposure to the bone marrow;harvesting a tissue or organ of the donor mammal; and determining thatthe tissue or organ of the donor mammal is resistant tocomplement-dependent cytotoxicity mediated by preformed antibodies ofthe sensitized recipient mammal reactive with donor antigen; wherein themethod does not include administering an accommodation-inducing factorother than the bone marrow.
 2. The method of claim 1, wherein the bonemarrow is from a mammal which is of the same species as the sensitizedrecipient.
 3. The method of claim 2, wherein said sensitized recipientmammal is a human.
 4. The method of claim 2, wherein the bone marrow isfrom the sensitized recipient mammal.
 5. The method of claim 1, whereinthe tissue or organ is heart, kidney, liver, lung, pancreas, heart-lung,intestine, spleen, or thymus.
 6. The method of claim 1, wherein thetissue is bone, skin, hair, eye, neural tissue, skeletal muscle, smoothmuscle, cardiac muscle, myocytes for skeletal muscle, myocytes forsmooth muscle, myocytes for cardiac muscle, pancreatic islets,hepatocytes, stem cells, progenitor cells, or hematopoietic cells. 7.The method of claim 1, wherein said step of determining occurs prior tothe step of harvesting.